Fluorescent dyes are widely used in biological assays (e.g., DNA and protein microarrays, DNA/RNA/Protein blotting, etc.), imaging (confocal, epifluorescence, pathology, live- and fixed-cell in vitro, whole-body in vivo, etc.) and diagnostics (e.g., in vitro diagnostics, sandwich assays, lateral flow assays). Enhancement of the signal strength of the fluorescent dye would be beneficial for the such uses of fluorescence, either for enhancing sensitivity (e.g., finding low-abundance target molecules) or increasing throughput (e.g., decreased integration time for imaging). The simplest approach to increasing signal strength is to increase the fluorophore concentration. However, this approach is generally not possible for conventional fluorescent dyes because the dye molecules interact electronically with each other and quench the signal at high concentrations. This is the case both for free dyes in solution as well as dyes bound to biomolecules or surfaces. Thus, any signal gain is offset by signal loss due to quenching at high enough local concentrations (e.g., multiple dyes on a single antibody). Another deficiency of traditional fluorescent dyes includes photobleaching, or photo-oxidation of the dye to a non-fluorescent form by the light source, which results in dye degradation and loss of fluorescent signal. Rates and modes of photobleaching are often affected by interactions with solvent molecules, such as water. Photobleaching limits dye longevity under illumination, which detrimentally affects the imaging or detection of the substrate when long integration times or images at multiple timepoints are required.
Therefore, as a result of these shortcomings of traditional fluorescent dyes, a need exists for new fluorescent dyes that can generate strong, long-lasting signals.